Alcohol drinking and certain pathophysiological conditions such as fasting and diabetes increase the levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and other P450 enzymes in humans and animal models. The substrates of CYP2E1 include: ethanol, acetaldehyde, acetaminophen (APAP), 4-hydroxynonenal, carbon tetrachloride, nitrosamines, and long chain fatty acid such as arachidonic acid (AA) and docosahexaenoic acid (DHA). Increased CYP2E1 leads to the production of more reactive metabolites of CYP2E1 substrates while reducing cellular anti-oxidants such as glutathione. The reactive metabolites include: acetaldehyde, reactive oxygen species, free radical metabolites and lipid peroxides. Therefore, cells or tissues with increased CYP2E1 become more susceptible to damage or cell death, especially in the presence of an additional factor working in a synergistic manner. However, the molecular signaling mechanisms for the toxicities caused by various CYP2E1 substrates have not been studied in detail. During the last three or four years, we have been studying the mechanism of cell damage caused by alcohol and other CYP2E1 substrates. Our initial hypotheses were that CYP2E1 substrates and their metabolites would activate the c-Jun N-terminal protein kinase (JNK) and p38 mitogen activated protein (MAP) kinase associated with the cell death pathway while they would suppress the enzymes involved in the cell survival pathway. Therefore, we specifically investigated the time-dependent activation and the role of the JNK and p38 kinase in cell death after exposure to CYP2E1 substrates. Recent results from this laboratory showed that three CYP2E1 substrates (APAP, 4-hydroxynonenal, and carbon tetrachloride) cause apoptosis through selective activation of the JNK-related cell death pathway. During this past year, we continued to investigate whether other CYP2E1 substrates such as DHA and ethanol cause cell damage in a similar mechanism. Our results showed that DHA causes time and dose-dependent apoptosis of HepG2 hepatoma cells (E47) transfected with CYP2E1 cDNA. In contrast, DHA causes significantly less damage to control HepG2 cells (C34) without CYP2E1 cDNA. DHA-induced damage to E47 HepG2 cells was followed by cytochrome c release and activation of caspase 3, a critical enzyme in executing apoptosis. Inhibition of CYP2E1 and JNK by chlormethiazole and quercetin, respectively, markedly reduce the rate of DHA-mediated damage to E47 cells, suggesting the important role of CYP2E1 metabolism of DHA and JNK activation in DHA-mediated cell death. Furthermore, the cell death rate did not correlate with the levels of lipid peroxides accumulated in E47 hepatoma cells. Our results, therefore, demonstrate that the early signaling event may be more important than the steady state levels of lipid peroxides in causing the cell death. In addition, we studied the mechanism of cell death caused by ethanol. Ethanol exposure caused cell death in HCT-116 colon cells and SK-N-SH neuroblastoma cells by activating the JNK and p38 kinases in both cells. Our preliminary results showed that selective inhibition of JNK or p38 kinase by their respective inhibitor markedly suppressed the rate of ethanol-induced cell damage in the colon cells, suggesting the importance of co-activation of JNK and p38-kinase mediated cell damage. This result is similar to the mechanism of cell death caused by other apoptotic stimuli such as hydrogen peroxide, UV and x-ray irradiations, and pro-inflammatory cytokines including tumor necrosis factor alpha, all of which activate p38 kinase along with the JNK in a coordinate fashion. Therefore, the mechanism of cell damage caused by various CYP2E1 substrates differ, depending on the type of target cells and each CYP2E1 substrate.